Solvent and kinetic isotope effects in the reaction of oxidative deamination of L-alanine, catalyzed by L-alanine dehydrogenase, AIaDH, （EC 1.4.1.1） were determined using a non-competitive spectroscopic method. The ...Solvent and kinetic isotope effects in the reaction of oxidative deamination of L-alanine, catalyzed by L-alanine dehydrogenase, AIaDH, （EC 1.4.1.1） were determined using a non-competitive spectroscopic method. The progress of the reaction was monitored spectrophotometrically by measuring the increasing absorbance of the reduced form of NADH at 340 nm. L-alanine, stereospecifically labeled with deuterium was synthesized by enzymatic reductive amination of pyruvate in presence of [（4R）-2H]-NADH, which was obtained by deuterium transfer from deuteriated formic acid to NAD~ catalyzed by FDH （formate dehydrogenase） （EC 1.2.1.2）. [2-2H]-L-alanine, the product of enzymatic synthesis catalyzed by AIaDH, was obtained with 75% deuterium enrichment and values of isotopic effects were approximated to the values corresponding to 100% of deuterium incorporation. The enzyme AIaDH isolated from Bacillus subtilis shows pro-R stereospecificity, what indicates that hydrogen is exclusively transferred from pro-R position at C-4 of the nicotinamide ring of NADH to C-2 of pyruvate to form L-alanine. Some intrinsic mechanistic details of enzymatic oxidative deamination of L-alanine were discussed using determined numerical values of kinetic and solvent isotope effects on Vmax and Vmax,│KM展开更多
文摘Solvent and kinetic isotope effects in the reaction of oxidative deamination of L-alanine, catalyzed by L-alanine dehydrogenase, AIaDH, （EC 1.4.1.1） were determined using a non-competitive spectroscopic method. The progress of the reaction was monitored spectrophotometrically by measuring the increasing absorbance of the reduced form of NADH at 340 nm. L-alanine, stereospecifically labeled with deuterium was synthesized by enzymatic reductive amination of pyruvate in presence of [（4R）-2H]-NADH, which was obtained by deuterium transfer from deuteriated formic acid to NAD~ catalyzed by FDH （formate dehydrogenase） （EC 1.2.1.2）. [2-2H]-L-alanine, the product of enzymatic synthesis catalyzed by AIaDH, was obtained with 75% deuterium enrichment and values of isotopic effects were approximated to the values corresponding to 100% of deuterium incorporation. The enzyme AIaDH isolated from Bacillus subtilis shows pro-R stereospecificity, what indicates that hydrogen is exclusively transferred from pro-R position at C-4 of the nicotinamide ring of NADH to C-2 of pyruvate to form L-alanine. Some intrinsic mechanistic details of enzymatic oxidative deamination of L-alanine were discussed using determined numerical values of kinetic and solvent isotope effects on Vmax and Vmax,│KM
